1. Field of the Invention
The present invention relates generally to methods and compositions for the enhancement of cellular proliferation and the treatment of wounds and other disorders. In particular, the invention relates to the use of defensin peptides for wound treatment and other applications.
Traumatic injury and disease can cause damage to the skin, tissue, and body organs which requires cellular regeneration for healing. Accidental injuries such as cuts, abrasions, burns, and intentional surgical procedures result in wounds which can affect large areas of the skin or affected body organs and can require lengthy periods to heal. Long healing times are a particular problem with wounds on sensitive areas, such as corneal wounds which are difficult to treat over prolonged periods. For these reasons, it would be desirable to provide methods and pharmacological agents which can be used to promote rapid healing of wounds and other injuries to the skin, tissue, and body organs.
A variety of cellular growth promoting hormones have been identified which can enhance cellular proliferation which have been used in wound treatment, including corneal wound treatment. Exemplary growth promoting hormones include epidermal growth factor, transforming growth factor .beta., insulin-like growth factor, platelet-derived growth factor, and the like. While use of these hormones continues to hold promise, no one growth promoting agent can be optimal for all situations. Moreover, it would be desirable to identify growth promoting agents which combine other desirable biologic activities, such as antimicrobial activity.
In addition to the wound itself, traumatic injury and surgical procedures present a substantial risk of microbial infection. While a wide variety of topical and systemic antimicrobial formulations are available, no one formulation is optimal for all circumstances. Moreover, it would be desirable to utilize antimicrobial compositions which possess additional desirable biological activities, such as the ability to promote cellular growth. It would also be desirable to identify antimicrobial compositions which are capable of inhibiting the growth of various resistant pathogens.
For the above reasons, it is an object of the present invention to provide pharmacological agents useful for the topical treatment of wounds and other disorders. Desirably, the compositions will be capable of providing a potent mitogenic activity which enhances the proliferation of epithelial cells, fibroblasts, and the like. The compositions will also be capable of inhibiting the growth of a wide variety of pathogenic and non-pathogenic microorganisms, including bacteria, viruses, and fungi. The compositions will be suitable for topical application to the skin and body organs, including the eye. Compositions will be suitable for incorporation into a wide variety of delivery vehicles.
2. Description of the Background Art
Defensins are a family of highly cross-linked, structurally homologous antimicrobial peptides found in the azurophil granules of polymorphonuclear leukocytes (PMN's) with homologous peptides being present in macrophages (Selsted et al., (1984) Infect. Immun. 45:150-154). Originally described as "Lysosomal Cationic peptides" in rabbit and guinea pig PMN Zeya et al., (1966), Science 154:1049-1051; Zeya et al., (1968), J. Exp. Med. 127:927-941; Zeya et al., (1971), Lab. Invest. 24:229-236; Selsted et al., (1984), supra.), this mixture was found to account for most of the microbicidal activity of the crude rabbit PMN extract against various microorganisms (Zeya et al., (1966), supra; Lehrer et al, (1977), J. Infect. Dis. 136:96-99; Lehrer et al., (1975), Infect. Immun. 11:1226-1234). Six rabbit neutrophil defensins have been individually purified and are designated NP-1, NP-2, NP-3A, NP-3B, NP-4, and NP-5. Their amino acid sequences were determined, and their broad spectra of activity were demonstrated against a number of bacteria (Selsted et al., (1984), Infect. Immun. 45:150-154), viruses (Lehrer et al., (1985), J. Virol. 54:467), and fungi (Selsted et al., (1985), Infect. Immun. 49:202-206; Segal et al., (1985), J. Infect. Dis. 151:890-894). Four peptides of the defensin family have been isolated from human PMN's and are designated HNP-1, HNP-2, HNP-3, and HNP-4 (Ganz et al., (1985), J. Clin. Invest. 76:1427-1435; Wilde et al. (1989) J. Biol. Chem. 264:11200-11203). The amino acid sequences of HNP-1, HNP-2, and HNP-3 differ from each other only in their amino terminal residues, while each of the human defensins are identical to the six rabbit peptides in 10 or 11 of their 29 to 30 residues. These are the same 10 or 11 residues that are shared by all six rabbit peptides. Human defensin peptides have been shown to share with the rabbit defensins a broad spectrum of antimicrobial activity against bacteria, fungi, and enveloped viruses (Ganz et al., (1985), supra.). Three defensins designated RatNP-1, RatNP-2, and RatNP-4, have been isolated from rat. Eisenhauer et al. (1989) Infection and Immunity 57:2021-2027. A guinea pig defensin (GPNP) has also been isolated, purified, sequenced and its broad spectrum antimicrobial properties verified (Selsted et al., (1987), Infect. Immun. 55:2281-2286). Eight of its 31 residues were among those invariant in six rabbit and three human defensin peptides. The sequence of GPNP also included three nonconservative substitutions in positions otherwise invariant in the human and rabbit peptides. Of the defensins tested in a quantitative assay HNP-1, RatNP-1, and rabbit NP-1 possess the most potent antimicrobial properties while NP-5 possesses the least amount of antimicrobial activity when tested against a panel of organisms in stationary growth phase (Selsted et al., (1984), Infect. Immun. 45:150-154; Ganz et al., (1985), J. Clin. Invest. 76:1427-1435). Defensin peptides are further described in U.S. Pat. Nos. 4,543,252; 4,659,692; and 4,705,777.
The use of growth factors in treating cutaneous and corneal wounds is described in European Patent Applications No. 190 019 and PCT Application No. WO 86/02271.
Work relating to the present invention was described in Murphy et al., (1989) Invest. Oph. Vis. Sci. Suppl., 30:149 and Mannis et al. (1989) Invest. Oph. Vis. Sci. Supp. 30:363.